Kits for determining sediment and pore water toxicity with dormant zooplankton having dormant life stage

ABSTRACT

The present disclosure provides methods for determining the toxicity of fresh-water and marine sediments and sediment pore water containing indigenous or introduced toxicants from each as a one-time analysis and/or for analysis over a period of time. The present disclosure further provides kits assembled for the afore-mentioned determination. The methods and kits of the present disclosure can be used for analyzing sediment and pore water samples from, among other locations, all environments where species having a dormant life stage may exist including, for example, natural zooplankton.

This application claims benefit of priority to provisional application62/169,832, filed Jun. 2, 2015.

SUMMARY

The present invention provides methods for determining the toxicity ofcertain indigenous or introduced toxicants in fresh-water and marinesediments, sediment pore water and terrestrial samples from each as aone-time analysis and/or for analysis over a period of time. The presentinvention further provides kits assembled for the afore-mentioneddetermination. The methods and kits of the present invention can be usedfor analyzing sediment and pore water samples from all environmentswhere natural zooplankton embryos or other species having a dormant lifestage exist or could exist under anoxic or other controlledenvironmental conditions.

BACKGROUND

The United Stated Environmental Protection agency and United States ArmyCorps of Engineers have been seeking a method for determining thechronic toxicity of fresh water and marine bottom sediment and porewater extracted therefrom. Although certain methods have been availablefor using invertebrate species for evaluating the toxicity of sedimentand pore water by introducing active or developing animals to thesediment and/or pore water, these tests are limited in duration due tothe physiological needs of the active invertebrates used. Moreover,there appears to be no known method that would provide for determiningthe toxicity of such sediment and/or pore water to dormant invertebratesover an extended period of time.

DETAILED DESCRIPTION Definitions

“Arthropod” has its common meaning as typically defined and used in thefield of biology, particularly as further described herein.

“Carried out over a period of time”, or variants thereof, means anyperiod of time other than a single point in time.

“Dormant” or “Dormancy” has it common meaning as typically defined inthe field of biology and includes, for example and without limitation,naturally occurring dormancy and induced dormancy via. Dormancy can beinduced, for example and without limitation via: i) naturally occurringform of dormancy called diapause that does not need to be induced butdoes need to be maintained via, for example, protection from light,dehydration, temperature changes and oxygen levels to maintain thediapause form of dormancy. ii) quiescence is a form of dormancy inducedby environmental conditions. Anoxia is the most common example. See theHand and Podrabsky article cited below as a reference for diapause andquiescence comparison. iii. eutrophication appears to be one cue toinduce dormancy in the freshwater jellyfish, as supported by theteachings of D E Culberson (1976) Studies of the encystment, excystmentand general ecology of Craspedacusta sowerbii. Masters of Sciencethesis, University of South Alabama, Auburn, Ala. (hereinafter,collectively “naturally occurring or induced dormancy”).

“Developmental deformity” means any deformation (development other thannormal development) of an emerging or hatching zooplankton, includingthe inability of such zooplankton to emerge or hatch. As used herein,“developmental deformity” can also include mortality as an evaluativeendpoint.

“Emerging” or “emerge” means protrusion or expulsion of a zooplanktonembryo or, more generally, a species having a dormant life stage, ordeveloping larva from its egg or cyst coat as an intermediate stage ofdevelopment prior to hatching

“Hatching” or “Hatch” means the release of a free-swimming zooplanktonlarva from any structure(s) that encapsulate the larvae during priordevelopment.

“Indigenous” means the presence of a toxicant in one or more sediment,or pore water sample without the addition of such toxicant to thesediment or pore water.

“Lipophilic” has its common meaning as typically defined and used in thefield of chemistry and includes, for example and without limitation, asubstance or chemical having any degree or amount of lipophilicity. Forfurther clarity, the term “lipophilic”, as used herein, includes anycompound or substance capable of penetrating the cuticle of an arthropodor other organism having a dormant state as further described herein.

“Osmolyte or Osmolytic Solution” has its traditional scientific meaningwith the caveat that when used herein, the osmolyte or osmolyticsolution does not penetrate the cuticle of a respective target speciessuch that lysis would occur or otherwise interfere with the action ofthe respective organism.

“Pore water” means the water extracted and/or separated from a sedimentsample.

“Reconstituted” means made by dissolving a specified amount of salts ofvarious kinds in a specified volume of water to create a standardizedsolution.

“Sediment” has its common meaning that includes terrestrial, aquaticfreshwater and aquatic marine environments. In addition, the term“sediment”, as used herein, also includes terrestrial soil samples usingthe methods and kits described herein and/or toxicant-contaminatedterrestrial soil that has been deposited and/or displaced to an aquaticfreshwater and/or aquatic marine environment.

“Species Having a Dormant Life Stage” means, typically, zooplankton asfurther described herein. Further examples of species having a dormantlife stage includes, for example and without limitation the followingdescriptions as set forth herein below:

-   -   Craspedacusta sowerbyii (Freshwater Jellyfish; podocyst is the        dormant life stage)        http://nas.er.usgs.gov/queries/FactSheet.aspx?speciesID=1068,        which is herein incorporated by reference in its entirety;    -   Acartia tonsa (marine copepod). F Chen and N H Marcus (1997)        Subitaneous, diapause, and delayed-hatching eggs of planktonic        copepods from the northern Gulf of Mexico: morphology and        hatching success. 127:587-597, which is herein incorporated by        reference in its entirety. This paper teaches that 6 species of        marine zooplankton from just one sampled location all produce        dormant embryos (Labidocera aestiva, Acartia tonsa, Centropages        velificatus and Calanopia americana), and two are capable of        producing both dormant and non-dormant embryos (Labidocera        mirabilis and Centropages hamatus);    -   Boeckella poppei and Boeckella triarticulata (freshwater/saline        lake copepods) X Jiang, S Zaho, Z Xu, G Wang, J He, and M        Cai (2012) Abundance and age of viable resting eggs of the        calanoid copepod Boeckella poppei Mrazek in sediments: evidence        of egg banks in two Antarctic maritime lakes. Polar Biology 35:        1525-1531, which is herein incorporated by reference in its        entirety. This article teaches that Boeckella poppei has a        dormant embryo;    -   K M Couch, M Downes and C W Burns (2001) Morphological        differences between subitaneous and di apause eggs of Boeckella        triarticulata (Copepoda: Calanoida). Freshwater Biology.        46:925-933, which is herein incorporated by reference in its        entirety. This article teaches the difference in structure        between embryos capable of dormancy and those that directly        develop for one species of copepod (B. triarticulata). A.        franciscana also has two types of embryos like this;    -   Diaptomus sanguineus (freshwater lake copepod). N G Hairston and        C M Kearns (2002) Temporal dispersal: ecological and        evolutionary aspects of zooplankton egg banks and the role of        sediment mixing. Integrative and Comparative Biology.        42:481-491, which is herein incorporated by reference in its        entirety. This paper teaches about the fact that natural        sediments maintain dormancy in egg banks of freshwater lakes,        and that removal from that environment by sediment mixing is        required to exit the dormant state. D. sanguineous is the        primary example species used;    -   Artemia monica (hypersaline lake anostracan). L E Drinkwater and        J H Crowe (1987) Regulation of embryonic diapause in        Artemia—environmental and physiological signals. Journal of        Experimental Zoology, 241:297-307, which is herein incorporated        by reference in its entirety;    -   Eunapius fragilis (freshwater sponge gemmules) and Artemi        franciscana (great salt lake and San Franscisco bay saltern        anostracan). S C Hand and J E Podrabsky (2000) Bioenergetics of        diapause and quiescence in aquatic animals. Thermochimica Acta.        349:31-42, which is herein incorporated by reference in its        entirety. This article teaches that diapause is an endogenously        programmed form of dormancy while quiescence is an        environmentally induced form of dormancy. The ordinarily skilled        artisan can either set conditions to maintain diapause or set        conditions to induce quiescence for this and other species        having a dormant life stage as described herein;    -   Artemia salina (hypersaline lake anostracan; mono lake) and        other Artemia species well known in the art; and    -   Branchinecta gainii (freshwater lake anostracan).

“Toxicant” means an anthropogenically released or produced material(including, for example, compound(s) and substance(s)) that is/areintroduced to a freshwater, marine or terrestrial environment andcollects or resides in the indigenous sediment that is or can be toxicto marine or fresh-water zooplankton and/or other species having adormant life stage. As used herein, the term “toxicant” typicallyincludes lipophilic compounds or substances, and can also include amaterial introduced to a freshwater, marine, terrestrial or pore watersample for the purposes of carrying out the processes described herein.

Description

One aspect of the present invention provides for a method of determiningthe toxicity of indigenous or introduced toxicants in sediment samplescomprising: mixing a plurality of dormant embryos of at least onezooplankton species or, more generally, a species having dormant lifestage with a sediment sample under anoxic conditions; hatching the atleast one species having a dormant life stage; and observing theembryos, prelarvae and larvae of the at least one species having adormant life stage for any developmental deformity and/or mortality.

Another aspect provides for the additional step of separating thespecies having a dormant life stage, frequently a zooplankton species,from the sediment by using a vessel in which the species having adormant life stage and sediment are mixed (a “mixing vessel”) prior toemergence and hatching testing.

An additional aspect provides for the additional steps of: mixing thesediment with a sucrose solution or other non-toxic osmolyte orosmolytic solution that in a vessel capable of being used in acentrifuge including for example and without limitation a mixing vessel;centrifuging the mixture from or in the mixing vessel; separating theembryos from the supernatant from the centrifuging by pouring thesupernatant over a sieve or filter paper, leaving the embryos in thesieve or filter paper; and transferring the embryos to another vesselfor observations related to, for example and without limitation,hatching, emergence, developmental deformity/deformities, mortality andother observations and/or tests described herein, including for exampleand without limitation the timing of such hatching, emergence anddevelopmental events or that can be carried out by the ordinarilyskilled artisan. The embryos can be optionally rinsed to wash off thesucrose or other osmolyte solution before the referenced transfer.

For primary or secondary school demonstrations or other instances when acentrifuge is not readily available, a string or monofilament line canbe secured to an anoxic or other respective incubation chamber asdescribed herein and swung at a high speed in a circular manner tosegregate some of the or, more generally, at least one species having adormant life stage, from a sediment sample. Alternatively, for exampleand without limitation, a salad spinner can be used to generate thenecessary centrifugal force needed to segregate species having a dormantlife stage from a sediment sample. Essentially, any device, commerciallyavailable or self-constructed, capable of providing sufficientcentrifugal force required to carry out the processes described hereinwould be acceptable. This method is not intended to be used forquantitative experimentation but may be useful for demonstrationpurposes. It is suggested that additional zooplankton embryos, and/orembryos of at least one species having a dormant life stage, beintroduced to the sediment samples to increase the likelihood ofsegregation of embryos from sediment. Appropriate safety measures needbe taken when using this method. It is also recommended that a plastictube be used for this segregation method.

Another aspect provides for a method of determining the toxicity ofindigenous or introduced toxicants in sediment samples comprising:mixing a plurality of dormant embryos of at least one species having adormant life stage with a sediment sample under conditions to maintainsuch dormancy; hatching the at least one species having a dormant lifestage; and observing the embryos, prelarvae and larvae of the at leastone species having a dormant life stage for any developmental deformityor, more generally, observing recovery from the dormant state andensuring events associated with the continuation of the life cycle ofthe organism.

The present invention further provides for carrying out the methodsdescribed above over a period of time. In this instance, the length ofthe period of time is limited only by the experimental design andresources required to carry out the processes described herein. Morespecifically, such period of time can be from 1 to each of 24 hours in aday with each hour in a day being a whole and/or fractional number, oneto each of seven days in a week with each day being a whole orfractional part of a day, from 1 to 52 weeks in a year with each weekbeing a whole or fractional week, from 1 to 12 months in a year witheach month being a whole or fractional month, from 1 to a plurality ofwhole or fractional years, and the like. In fact, the amount of time andfrequency of sampling sediment or pore water is not limited in time orfrequency of observation, depending upon the availability of samplematerials and materials to carry out the methods and use of the kits asdescribed herein.

When studies are carried out over a period of time, sediment and/or porewater toxicant(s) can be supplemented to better understand the level(s)of indigenous toxicant versus a known quantity relative to the hatching,emergence and development of the zooplankton species or, more generally,species having a dormant life stage. Accordingly, another aspect of thepresent invention provides a method of comparing indigenous sediment andpore water toxicity relative to known toxicants comprising: adding aknown quantity of at least one known toxicant to at least one sedimentor pore water sample; and comparing the hatching, emergence anddevelopment of the respective zooplankton species or, more generally,species having a dormant life stage, between the sample(s) containingindigenous toxicant versus samples containing supplemented toxicant.

Another aspect of the present invention provides a method fordetermining indigenous or introduced toxicants in pore water collectedfrom sediment samples comprising: mixing a plurality of dormant embryosof at least one species having a dormant life stage with a the porewater sample under anoxic conditions or other conditions that willinduce or maintain dormancy; hatching the at least one species having adormant life stage; and observing the emerging embryos, prelarvae orlarvae of the at least one species having a dormant life stage for anydevelopmental deformity and/or mortality.

Another aspect provides for a method of determining the toxicity ofindigenous or introduced toxicants in pore water samples comprising:mixing a plurality of dormant embryos of at least one species having adormant life stage with a pore water sample under conditions to maintainsuch dormancy; hatching the at least one species having a dormant lifestage; and observing the embryos, prelarvae and larvae of the at leastone species having a dormant life stage for any developmental deformityor mortality or, more generally, observing recovery from the dormantstate and ensuring events associated with the continuation of the lifecycle of the organism.

An additional aspect provides for the additional steps for the porewater processes taught herein above of: separating the embryos from thepore water by pouring the pore water containing the at least onezooplankton species or, more generally, at least one species having adormant life stage, over a sieve or filter paper leaving the embryos inthe sieve or filter paper; and transferring the embryos to anothervessel for observations related to, for example and without limitation,hatching, emergence, developmental deformity/deformities, mortality andother observations and/or tests described herein or that can be carriedout by the ordinarily skilled artisan. These methods can also be carriedout over a period of time.

Also provided is a kit for determining indigenous or introducedtoxicants in sediment samples comprising: a plurality of embryos of atleast one zooplankton species or, more generally, at least one specieshaving a dormant life stage; at least one dry water-preparation packageselected from the group consisting of artificial (“reconstituted”)freshwater mix and artificial seawater mix (each, individually, a “drysalt package”); at least one anoxic incubation container that can bepurged; and a sterile receptacle for emergence and hatching testing ofthe zooplankton or, more generally, species having a dormant life stage.In another aspect, the anoxic incubation chamber can be replaced withvessels known to the ordinarily skilled artisan in which dormancy of thespecies having a dormant stage is maintained.

Another aspect provides for the addition of material to theabove-described kit comprising: at least one selected from the groupconsisting of a squirt bottle, sieve or filter material, dry sucrosecrystals or other non-toxic osmolyte or osmolytic solution, instructionsincluding diagrams of developing or other species having a dormant lifestage and tubes capable of being used in a centrifuge or other means bywhich to separate or segregate the species having a dormant life stagefrom sediment.

An additional aspect of the present invention provides for a kit fordetermining the toxicity of indigenous or introduced toxicants insediment pore water samples comprising: a plurality of embryos of atleast one zooplankton species or, more generally, at least one specieshaving a dormant life stage; at least one dry salt package selected fromthe group consisting of artificial freshwater mix and artificialseawater mix; at least one anoxic incubation container that can bepurged or other appropriate container in which dormancy of a specieshaving a dormant life stage is induced or maintained ; and a sterilereceptacle for emergence and hatching testing of the zooplankton or,more generally, species having a dormant life stage, optionally alsocomprising at least one selected from the group consisting of a squirtbottle, instructions including diagrams of developing zooplankton or,more generally, species having a dormant life stage, and sieve or filtermaterial; the squirt bottle and sieve or filter material being wellrecognized in the chemical and biological arts.

Sediment samples can be taken by any method. Typically, but withoutlimitation, core or grab sample is taken to any desired depth foraquatic or terrestrial samples. The sediment sample can be incrementallysegregated by depth or can be mixed to prepare a single homogeneoussediment sample. Sediment should be maintained under normoxic, hypoxicor anoxic conditions that most closely approximate conditions at thesite of origin.

A variety of zooplankton embryos or, more generally, at least onespecies having a dormant life stage, can be used to carry out themethods of the present invention and can be included in the kitsdescribed herein. Typically, embryos of one or more such species is/areselected to match the environment from which a respective sedimentsample is taken. For example, a species having a dormant life stageindigenous to fresh-water should be used when testing fresh-watersediment samples or samples to be added to a freshwater system and aspecies having a dormant life stage indigenous to a marine environmentshould be used when testing marine sediment samples or samples to beadded to a marine system.

For example and without limitation, embryos of the salt-water (marineand hypersaline) species Artemia franciscana can be used for testingmarine sediment and/or pore samples or used in the kits of the presentinvention. Typically this Artemia species is used/provided in a kit in adechorionated and hydrated form. For each of the methods and kits of thepresent invention, the use of Artemia species that are known for high,consistent hatch rates are beneficial. One supplier of such zooplanktonis the Great Salt Lake Brine Shrimp Cooperative (Ogden, Utah, USA). Forthe sake of clarity, a reference to zooplankton herein includes, forexample and without limitation, references herein to any Artemiaspecies. For species described and used in the methods and kits herein,the ordinarily skilled artisan will appreciate that varying methods areavailable for accomplishing dechorionation, as appropriate for eachselected species.

Useful fresh-water embryonic zooplankton species include redtail fairyshrimp (also, typically, dechorionated and hydrated), available from,for example, Arizona Fairy Shrimp (http://arizonafairyshrimp.com) andFlorida Aquafarms (http://florida-aqua-farms.com/). Additionalzooplankton embryos that can be used in the present methods and kitsinclude rotifers, Daphnia spp., Triops spp and other culturedzooplankton that are sensitive to lipophilic toxicants. Such zooplanktonembryos are readily available from a variety of aquaculture suppliers.

Marine and/or fresh-water embryonic zooplankton species can be used forterrestrial soil samples depending upon the closest potentialenvironment to such samples and/or of the experimental design of theresearcher. It may be optimal to test both fresh-water and marinezooplankton species in terrestrial samples as one would not be limitedto the indigenous environment providing a proper environment, asdescribed herein, is created for the study of introduced zooplanktonspecies. Additional species having a dormant life stage are additionallydescribed hereinabove.

Depending upon the species of species having a dormant life stage usedin the methods and kits of the present invention, dehydrated embryos(those not pre-hydrated) need to be hydrated for about 4 to about 24hours, typically on ice prior to use. In some cases, embryos of certainspecies like Daphnia magna should be stored hydrated at 4° C., and onlyused after they are stored for at least 1 month.

With the present methods and for use with the kits described herein, theselected species having a dormant life stage embryos are mixed with thesediment and/or pore water sample(s) to be analyzed. Embryo hatch andconformity or deformity or lack of emergence or hatching and mortality,if present, can be determined directly with the sediment by transferringa sample of the sediment and/or pore water to another container,typically a sterile petri dish or a plurality of independent orinter-connected vessels, the latter typically being multi-well, sterilepolystyrene culture plates. Twelve-well polystyrene culture plates workwell when simultaneously testing multiple sediment or pore watersamples. It may be beneficial to segregate sediment samples into anumber of containers equal to the number of tests to be conducted priorto introducing the desired species having a dormant life stage embryointo such container.

Initially, dormant species having a dormant life stage, particularlyzooplankton embryos, (supplied in the kit described herein) are mixed inwith one or more sediment samples in a hatching medium, frequently in atreatment flask. The number of embryos used for each treatment to beanalyzed depends upon whether the intent of the test is a singlepoint-in-time test or tests to be run over a period of time and whetherthe tests are to be replicated for statistical analysis. Generally, 10to 30 embryos per ml of hatching medium, or at least 100 embryos intotal are used per individual, un-replicated test. The hatching mediumcan contain ordinary water, distilled water, deionized water and thelike. Typically, a freshwater (“reconstituted”) hatching medium isprepared using deionized water with an artificial water mix as prepared,for example, by Kluttgen (Kluttgen, B, Dulmer, U, Engles, M, Ratte, H.T., 1994. ADaM, an artificial freshwater for the culture of zooplankton.Water Res. 28, 743-746), which is herein incorporated by reference inits entirety. For some species of zooplankton, a dilute solution ofartificial seawater with a salinity of 0.1 to 0.5 parts per thousand(ppt) can be used in place of an artificial freshwater mix. In thiscase, salinity is determined using methods well known in the art.

When required, an artificial seawater mix is prepared such that the saltcontent is about 20 ppt. Artificial seawater is commercially availablefrom numerous outlets but, for example, can also be prepared via themethod taught by Neumeyer, et al. (Neumeyer, C. H., Gerlach, J. L.,Ruggerio, K. M., and Covi, J. A., 2015. A novel model of earlydevelopment in the brine shrimp, Artemia franciscana, and its use inassessing the effects of environmental variables on development,emergence, and hatching. Journal of Morphology: 276, 342-360, which isherein incorporated by reference in its entirety).

To maintain exposure of introduced species having a dormant life stage,particularly zooplankton, over a period of time and to prevent thehatching of the selected species, it is best to maintain sediment andpore water under anoxic conditions or other conditions that will induceor maintain dormancy. Maintaining samples under anoxic or otherappropriate conditions can be carried out by methods well known to theskilled artisan. One option is to place samples in small vials,typically glass vials, and purge the vials with an inert gas primarily,without limitation, nitrogen gas. Alternatively, glass vesselspre-fitted with a purging cap for purging with inert gas are readilyavailable on the commercial market (see, e.g., Foxx Life Sciences,Salem, N H). The vessels described herein can be used as theafore-mentioned treatment flask wherein the sediment or pore watersamples containing introduced species having a dormant life stage aremaintained under anoxic or other such appropriate conditions for thedesired length of time prior to hatching and observation.

A single point test can be conducted by removing the anoxic conditionsor other conditions used to maintain dormancy species having a dormantlife stage in a sediment sample, transferring an aliquot of the sedimentand observing the hatching of the introduced species having a dormantlife stage, particularly zooplankton, generally, under a microscope andtypically under a dissecting microscope with 75× or greater maximummagnification.

For the purpose of evaluating the impact of toxicants on earlydevelopment of species having a dormant life stage, particularlyzooplankton, it is best to separate the introduced embryos from arespective sediment sample and observe individual embryos under amicroscope. Separation of embryos from sediment can be carried out via avariety of processes. Generally, water from a treatment flask isdecanted and sediment is mixed with a sucrose solution or othernon-toxic osmolyte or osmolytic solution that does not penetrate thecuticle of a respective target species in a vessel capable of beingcentrifuged. When using a sucrose solution, the concentration of thesucrose solution is typically a concentration selected from the groupconsisting of at least 10% sucrose, at least 20% sucrose, at least 30%sucrose, at least 40% sucrose, at least 50% sucrose, at. least 60%sucrose, at least 70% sucrose, at least 80% sucrose and at least 90%sucrose. Typically, a disposable 50 mL or 15 mL conical vial is useful.The mixture in the vial is then centrifuged until the sediment isseparated from the remaining fluid. It has been found that centrifugingthis mixture at about 1,000 rcf (relative centrifugal force) for aboutone minute is sufficient to separate the sediment from the selectedspecies-containing supernatant.

Following centrifugation, the supernatant is poured over an appropriatefilter device, typically a sieve, filter cloth or filter paper, tocollect the embryos, the embryos optionally can be washed to remove anyremaining sugar or other osmolyte, and the embryos are then transferredto an appropriate vessel or plurality of vessels from which observationsare made via microscope. Observation in a sterile petri dish is onealternative. For multiple observations where individual or small groupsof 2 to 100 embryos are examined repeatedly over a period of time,filtered embryos can be transferred into a plurality of separate orinter-connected vessels (each, a hatching vessel); the latter including,for example and without limitation, sterile, 12-well polystyrene cultureplates.

For embryo hatch/deformity/mortality observations, an adequate number ofembryos are added to each vessel (as determined by a respectiveexperimental design. Generally, 100 or more embryos per experimentaltreatment group are an adequate number to generate reliable resultsrelative to emerging or hatching ratios (the relative number of emergedor hatched embryos over the total number of embryos added to arespective emergence or hatching vessel) and any developmentaldeformities. To facilitate the development, emergence and hatching ofone or more respective species having a dormant life stage, it isrecommended that an appropriate aliquot of the afore-mentioned water(fresh or marine for a respective species), also referred to as ahatching medium, be added to each hatching vessel. Generally, 1 mL ofsuch hatching medium in each well of a 12-well culture plate issufficient to aid in the hatching of the afore-mentioned number ofembryos. The number of embryos and the amount of hatching medium isdetermined by the experimental design and experience of the user of themethods and/or kits of the present invention.

Pore water samples are treated similarly to the above-described methodsfor sediment sample except there is no need to separate the introducedembryos from sediment, as none should be present in pore water samples.If sediment does exist in pore water samples, the above-describedprocedure can be used to separate embryos from such sediment.

Generally, pore water can be separated from sediment samples under avacuum or other means. Appropriate embryos are introduced to each porewater sample that is then maintained under anoxic or other appropriateconditions as described hereinabove. Depending upon experimental design,hatch/deformity/mortality tests can be conducted directly in pore wateras a single point test by removing the anoxic or other conditions usedto induce or maintain dormancy and transferring a sample ofembryo-containing pore water to one of the hatching vessels describedabove and the embryos observed accordingly.

Alternatively, embryos introduced to pore water, following the desiredexposure time, can be filtered and processed for observation asdescribed hereinabove.

Once embryos are transferred to a hatching vessel, typically containinga hatching medium, observation intervals can vary among species. Toobserve development, emergence and hatching and any embryo, prelarval orlarval deformation, it is recommended, for example, to observe Artemiafranciscana at least at two-hour intervals between eight and 20 hoursafter initiation of the observations/tests described herein at 22° C.Longer intervals are possible at colder temperatures as low as 15° C.,and shorter intervals are possible at warmer temperatures as high as 28°C. Observation interval times may vary among species and experimentaldesign intended endpoints.

Also provided herein are kits for carrying out the methods describedherein.

One such kit for determining indigenous or introduced toxicants insediment samples comprises: a plurality of embryos of at least onespecies having a dormant life stage, particularly a zooplankton species;at least one dry salt package selected from the group consisting ofartificial freshwater mix and artificial seawater mix; at least oneanoxic incubation container that can be purged; and a sterile receptaclefor hatching the zooplankton or other species having a dormant lifestage. In lieu of the anoxic chamber, another vessel appropriate forinducing and/or maintaining dormancy may be provided. This kit mayfurther comprise at least one selected from the group consisting of asquirt bottle, sieve or filter material, dry sucrose crystals or otherosmolyte, instructions including diagrams of developing zooplankton orother species having a dormant life stage and tubes capable of beingused in a centrifuge or other means by which to separate or segregatethe species having a dormant life stage from sediment.

The present invention also provides a kit for determining toxicity ofindigenous or introduced toxicants in sediment pore water samplescomprising: a plurality of embryos of at least one artificial freshwatermix and artificial seawater mix; at least one anoxic incubationcontainer that can be purged; and a sterile receptacle for hatching theor other species having a dormant life stage. In lieu of the anoxicchamber, another vessel appropriate for inducing and/or maintainingdormancy may be provided. This kit may further comprise at least oneselected from the group consisting of a squirt bottle and sieve orfilter material.

Kits designed for sediment samples may also be readily used for testingpore water samples.

The following examples are intended only for the purpose ofexemplification and are not intended to limit the scope or teachings ofthe instant application in any manner whatsoever.

EXAMPLES Example 1 Overview of Kits

-   -   i) Preparation—Depending upon the species, embryos are shipped        dry and aerobic (all species, unless dechoriated/decapsulated)        or hydrated and in a dormant state produced by anoxic or other        conditions. If hydrated, the end user requires no preparation.        If dehydrated, embryos may be shipped with a desiccant pack to        maintain dry conditions. If dehydrated, embryos are rehydrated,        typically with deionized water, for 4-24 hours on ice before        use. In some cases, embryos of certain species like Daphnia        magna are stored hydrated at 4° C., and only used after they are        stored for at least 1 month.    -   ii) Primary Treatment—A plurality of at least one zooplankton or        other species having a dormant life stage species are mixed with        field-collected sediment and/or field-collected pore water under        anoxic conditions. The number of zooplankton or other species        having a dormant life stage embryos, or dormant equivalent,        mixed with each sediment or pore water sample is as described        herein.    -   iii) Separation:        -   a) Hatching from sediment—The end user can test for larval            deformation without separating embryos from the sediment or            pore water samples by transferring the sediment and/or pore            water to a petri dish or other container that is compatible            with the working distance of a dissecting microscope.        -   b) Separating from sediment—To evaluate the impact of            sediment and toxicants on zooplankton early development, the            embryos are separated from sediment by pouring off water            from a treatment flask as described above and sediment is            mixed at ratios of 10:1 to 1:1 for sediment to sucrose with            an eighty percent (80%) sucrose solution in a disposable 50            mL or 15 mL conical vial. The mixture is then centrifuged at            1,000 rcf for one minute. Sediment particles are collected            at the bottom of the vial and the embryos float in the            supernatant that is poured over a sieve or filter paper for            collection of the embryos. The embryos are rinsed with            water, typically deionized water to remove any remaining            sugar, and transferred to one or more culture plates under            aerobic conditions for hatching and observation. For pore            water samples, such samples are poured over filter paper,            the zooplankton or other species having a dormant life stage            are collected and transferred to one or more culture plates            for observation during development, emergence and hatching.    -   iv) Emergence, Hatching and Developmental Observations—For        zooplankton or other species having a dormant life stage        separated from sediment or pore water, culture plates, for        example and without limitation, 12-well polystyrene culture        plates are prepared by placing 1 mL of an appropriate hatching        medium (artificial “reconstituted” freshwater or artificial        seawater as described herein) into each of the 12 wells.        Depending upon the end user's experimental design, an        appropriate number of the selected at least one or other species        having a dormant life stage species is placed in each well.        Typically, 10 to 30 embryos are transferred to each well.        Observations of development, emergence and hatching of the        zooplankton or other species having a dormant life stage are        conducted according to experimental design and vary according to        selected species. For example, observations are recommended at        about 2-hour intervals for Artemia franciscana. A developmental        flow diagram provided by the herein incorporated Neumeyer, et        al. article is used for continuous monitoring and endpoint        development, emergence and hatching values and an assessment of        developmental progression and timing. Accordingly, experimental        design using the methods and kits described herein permit        qualitative evaluations for the effects of the above-described        sediment and pore water samples with indigenous or supplemented        toxicants over any desired length of time.

Example 2

Sediment and Pore Water Toxicity Evaluation Kit when Testing Directlyfrom Sediment or Pore Water. Required and optional list items are listedbelow:

-   -   Dormant zooplankton or other species having a dormant life stage        (a plurality of at least one species)    -   Dry Salt pack—one or both of an freshwater mix and/or a salt        water mix    -   One or a plurality of anoxic incubation containers or other        containers for use to induce or maintain dormancy    -   One or a plurality of culture plates (one or more sterile petri        dishes or one or more multi-well, interconnected plates).    -   Plastic transfer pipette or pipettes

Example 3

Sediment and Pore Water Toxicity Evaluation Kit when Testing EmbryosSeparated from Sediment or Pore Water for detailed Observation. Requiredand optional items are listed below:

-   -   Dormant zooplankton or other species having a dormant life stage        (a plurality of at least one species)    -   Dry sucrose crystals or other osmolyte    -   Dry Salt pack—one or both of an freshwater mix and/or a salt        water mix    -   One or a plurality of anoxic incubation containers or other        containers used to induce or maintain dormancy    -   One or a plurality of culture plates (one or more sterile petri        dishes or one or more multi-well, interconnected plates)    -   One or a plurality of conical tubes for centrifugation    -   One or a plurality of plastic squirt bottles    -   One or a plurality of sieves, filter cloth or filter papers.    -   One or a plurality of plastic pipettes    -   One or a plurality of spatulas.

Example 4

-   -   Instructions for using the kits described herein comprise, for        example, the teachings for using kits as set forth herein and        are very broad to describe the broad range of tests that can be        carried out according to the teachings contained herein or are        specific for specific end-user needs such as, for example and        without limitation, instructions for using the kits for sediment        or pore water collected from fresh-water or marine environments.

Example 5

Items typically not, but may be, provided in Kit:

-   -   Inert gas, gas regulator and hose for purging anoxic incubation        containers    -   Deionized water    -   Centrifuge    -   Dissecting microscope    -   String, monofilament line or salad spinner    -   Conductivity meter or refractometer    -   Balance for determining mass.

The invention claimed is:
 1. A kit for determining the toxicity of sediment samples containing indigenous or supplemented toxicants comprising: a plurality of embryos of at least one species having a dormant life stage; at least one dry salt package selected from the group consisting of artificial freshwater mix and artificial seawater mix; at least one incubation container used to induce or maintain dormancy of the species having a dormant life stage; and a sterile receptacle for hatching the species having a dormant life stage.
 2. The kit according to claim 1, further comprising at least one selected from the group consisting of a squirt bottle, sieve or filter material, pipettes or spatulas, at least one osmolyte, instructions, and tubes capable of being used in a centrifuge.
 3. A kit for determining the toxicity of sediment pore water containing indigenous or supplemented toxicants comprising: a plurality of embryos of at least one species having a dormant life stage; at least one dry salt package selected from the group consisting of artificial fresh water mix and artificial seawater mix; at least one container used to induce or maintain dormancy of the at least one species having a dormant life stage; and a sterile receptacle for hatching the at least one species having a dormant life stage.
 4. The kit of claim 3, further comprising at least one selected from the group consisting of a squirt bottle, instructions and filter material. 